Protein Research
1. Protein identification: One-dimensional electrophoresis and two-dimensional electrophoresis combined with Western and other technologies can be used to identify proteins by using protein chips, antibody chips and co-immunoprecipitation technologies.
2. Post-translational modification: Many proteins produced by mRNA expression undergo post-translational modifications such as phosphorylation, glycosylation, and zymogen activation. Post-translational modification is an important way of protein regulation, so the study of protein post-translational modification plays an important role in elucidating the function of protein.
3. Determination of protein function: such as analysis of enzyme activity and determination of enzyme substrate, biological analysis of cytokines/ligand-receptor binding analysis. The function of gene expression products-proteins can be analyzed using gene knockout and antisense technologies. In addition, the study of the localization of the protein in the cell after the expression is also helpful to understand the function of the protein to a certain extent. Clontech’s fluorescent protein expression system is an excellent tool for studying the localization of proteins in cells.
4. For human beings, the research of proteomics should ultimately serve the health of human beings, mainly to promote the development of molecular medicine. Such as the search for drug target molecules. Many drugs are proteins themselves, and the target molecules of many drugs are also proteins. Drugs can also interfere with protein-protein interactions.
In basic medicine and disease mechanism research, it is of great significance to understand the characteristics of gene expression in different human development, growth stages, and under different physiological and pathological conditions and in different cell types. These studies may find molecules directly related to specific physiological or pathological states, further laying the foundation for designing drugs that act on specific target molecules.
Cellular Subcellular
The gene expression of different cell types is inconsistent in different developmental, growth stages and under different physiological and pathological conditions, so the study of protein expression should be precise to the cellular or even subcellular level. Immunohistochemical techniques can be used for this purpose, but the fatal disadvantage of this technique is its low throughput. Laser Capture Microdissection LCM (Laser Capture Microdissection) technology can precisely remove the cell types of interest to researchers from tissue sections, so LCM technology is actually an in situ technology. The removed cells are used for the preparation of protein samples. Combined with the technical route of antibody chip or two-dimensional electrophoresis-mass spectrometry, in situ high-throughput research on protein expression can be carried out. Many studies use the technical route of homogenizing tissue to prepare protein samples, and their conclusions are questionable, because the proteins of different cell types are mixed together after tissue homogenization, and the final research data cannot explain the expression of proteins in each type of cells. . Although a single type of cell can be obtained by culturing cells, it is difficult for cells cultured in vitro to simulate the environment of cells in vivo, so the conclusions drawn from such studies are difficult to explain the actual situation in vivo. Therefore, different cell types should be isolated first in research, and the isolated cells can be used for gene expression studies, including mRNA and protein expression.
Cells obtained by the LCM technique can be used for the preparation of protein samples. Total protein, or membrane protein, or nucleoprotein, etc., can be prepared as needed, and glycoproteins can also be enriched, or the complexity of protein types can be reduced by removing albumin. The relevant kits are provided by the manufacturer.
Two-dimensional Electrophoresis
Different types of proteins in a protein sample can be separated by two-dimensional electrophoresis. Two-dimensional electrophoresis can separate different kinds of proteins with high resolution according to their isoelectric points and molecular weight differences. Successful 2D electrophoresis can separate 2,000 to 3,000 proteins. After electrophoresis, the gel is stained with high sensitivity such as silver staining and fluorescent staining. If you want to compare the similarities and differences of protein expression between two samples, you can prepare the protein samples of the two under the same conditions, and then perform two-dimensional electrophoresis under the same conditions, and compare the two gels after staining. The two protein samples can also be labeled with different fluorescent dyes, and then the two protein samples are separated by two-dimensional electrophoresis on a piece of gel, and finally the results are analyzed by fluorescence scanning technology.
After the gel is stained, the gel image analysis system can be used to image, and then the protein spots can be quantitatively analyzed by the analysis software, and the protein spots of interest can be located. Through a special protein spot cutting system, the glue area where the protein spots are located can be precisely cut. Then, the protein in the gel is digested by enzyme digestion, and the digested digested product can be spotted on the surface of a specific material through a spotting system after desalting/concentration treatment (MALDI-TOF). Finally, these proteins can be analyzed in a mass spectrometry system to obtain qualitative data of the protein; these data can be used to construct a database or conduct a comparative analysis with an existing database.
The technical route of LCM-two-dimensional electrophoresis-mass spectrometry is a typical technical route of proteomics research. Besides, LCM-antibody chip is also an important technical route of proteomics research. That is, the cell types of interest are obtained by LCM technology, and cell protein samples are prepared. After the proteins are labeled with fluorescent dyes, they are hybridized with the antibody chip, so that the similarities and differences of protein expression between the two samples can be compared. Clontech recently developed an antibody chip that can analyze 378 membrane and cytoplasmic proteins. The chip is also equipped with important reagents for the entire operation process of the antibody chip, including protein preparation reagents, fluorescent dye labeling reagents for proteins, purification reagents for labeling systems, hybridization reagents, and the like.
For the study of protein interactions, yeast two-hybrid and phage display technology are undoubtedly good research methods. The yeast two-hybrid system developed by Clontech and the phage display technology developed by NEB are available to researchers.
For the study of the proteome, part or all of the proteins in the proteome can also be made into protein chips, which can be used for protein interaction studies, protein expression studies and small molecule protein binding studies. Science, Vol. 293, Issue 5537, 2101-2105, September 14, 2001 published a paper on yeast proteome chips. The main research content of this paper is: expressing 5800 ORFs of yeast into proteins, purifying and spotting to make a chip, and then using the chip to screen the interaction molecules between calmodulin and phospholipid molecules.
Finally, it is necessary to point out that traditional protein research focuses on the study of a single protein, while proteomics focuses on the study of all protein species involved in a specific physiological or pathological state and their relationship with the surrounding environment (molecules). Therefore, proteomic studies are usually high-throughput. To meet this requirement, proteomics-related research tools are usually highly automated systems with high throughput and fast speed. With corresponding analysis software and databases, researchers can process the most data in the shortest time.
